We will establish general methods to determine the assembly pathways of viruses and self- assembling nanoparticles. We will demonstrate these methods using Hepatitis B virus (HBV) capsid assembly. Resistive-pulse sensing allows real time monitoring of assembly by identifying single particles and assembly intermediates. Understanding the mechanism of virus assembly requires not only knowledge of precursors and final product structures, but also access to intermediates. Where many rare intermediates are involved, ensemble methods obscure them so that virus assembly resembles a two-state reaction. HBV has acutely infected more than 2B people; about 360M people have chronic HBV; every year nearly 1M will die of HBV-related liver disease. Assembly of HBV's icosahedral capsid has been identified as a new target for antiviral therapies. HBV assembly and the behavior (and development) of antiviral assembly effectors are relatively well understood, largely stemming from work in the Zlotnick lab, but there has been no direct observation and characterization of critical early intermediates in solution. Computational models of assembly suggest that the observed kinetics reflect early establishment of a constellation of intermediates needed to support capsid formation. The nucleation step and early intermediates are believed to play a role in recruiting viral components in vivo. Antiviral assembly effectors over-stimulate nucleation, distorting the distribution of intermediates and very often their structure. In our previous work, we established HBV assembly as a well-defined experimental system for nanofluidics and now have the foundation to interrogate assembly and antiviral assembly effectors. Nanofluidic components integrated with microfluidic devices offer a unique platform for answering these outstanding questions. Resistive-pulse sensing on these devices permits a real time, label-free approach to monitoring assembly at biologically relevant concentrations (nM to mM). More specifically, we will develop devices and methods to study particle transport properties through nanochannel networks. These coupled nanochannels can be arranged in virtually any two-dimensional format and operated with modest applied potentials. How particle transport is influenced depends strongly on the dimensions and geometries of the nanoscale conduits, applied waveforms, surface properties of the conduit, and composition of the transport medium, and particle shape and composition. We will optimize these device parameters in order to develop a fundamental understanding of capsid formation. The Specific Aims for this application are to: (1) characterize capsid assembly under various reaction conditions; (2) fabricate and test in-plane nanochannels with single pores and multiple pores in series for improved resistive-pulse sensing; (3) computationally simulate (i) assembly and (ii) particle transport in nanofluidic devices; and (4) develop coupled nanochannels to sense particles of different sizes and to perform reactions with single capsids.